This invention relates to the design and construction of amplification oligonucleotides and probes to Human Immunodeficiency Virus Type 1 (HIV), which allow detection of the organism in a test sample.
This section provides a brief outline of relevant areas. None of the art cited or referred to is admitted to be prior art to the claims. Laboratory diagnosis of Human Immunodeficiency Virus Type 1 in humans is currently performed by demonstration of the presence of viral antigen (p24) or anti-HIV-1 antibodies in serum. Direct detection of viral DNA, however, is a more useful diagnostic tool in some populations, such as infants born to seropositive mothers. Detection of viral DNA is more rapid and less hazardous than culture. Direct hybridization lacks adequate sensitivity in most patients (Shaw et al. Science 226:1165-1171, 1984). Many references mention oligonucleotides said to have use in detection of Human Immunodeficiency Virus. Most of these references also mention the use of polymerase chain reaction (PCR). These references include the following: Kwok et al., J. Virol. 61: 1690-1694, 1987; Agius et al., J. Virol. Meth., 30:141-150, 1990; Albert and Fenyo, J. Clin. Microbiol. 28:1560-1564, 1990; Bell and Ratner, AIDS Res. and Human Retroviruses 5:87-95, 1989; Bruisten et al., Vox Sane 61:24-29, 1991; Clarke et al., AIDS 4:1133-1136, 1990; Coutlee et al., Anal. Biochem. 181:96-105, 1989; Dahien et al., J. Clin. Microbiol. 29:798-804, 1991; Dudding et al., Biochem. Biophys. Res. Comm. 167:244-250, 1990; Ferrer-Le-Coeur et al., Thrombosis and Haemostasis 65:478-482, 1991; Goswami et al., AIDS 5:797-803, 1991; Grankvist et al., AIDS 5:575-578, 1991; Guatelli et al., J. Virol. 64:4093-4098, 1990; Hart et al., Lancet 2 (8611):596-599, 1988; Holland et al., Proc. Natl. Acad. Sci. USA, 88:7276-7278, 1991; Keller et al., Anal. Biochem. 177:27-32, 1989; Kumar et al., AIDS Res. and Human Retroviruses 5:345-354, 1989; Linz et al., J. Clin. Chem. Clin. Biochem. 28:5-13, 1990; Mano and Chermann, Res. Virol. 142:95-104, 1991; Mariotti et al., AIDS 4:633-637, 1990; Mariotti et al., Transfusion 30:704-706, 1990; Meyerhans et al., Cell 58:901-910, 1989; Mousset et al., AIDS 4:1225-1230, 1990; Ou et al., Science 239:295-297, 1988; Pang et al., Nature 343:85-89, 1990; Paterlini et al., J. Med. Virol. 30:53-57, 1990; Perrin et al., Blood 76:641-645, 1990; Preston et al., J. Virol. Meth. 33:383-390, 1991; Pritchard and Stefano, Ann. Biol. Clin. 48:492-497, 1990; Rudin et al., Eur. J. Clin. Microbiol. Infect. Dis. 10:146-156, 1991; Shoebndge et al., AIDS 5:221-224, 1991; Stevenson et al., J. Virol. 64:3792-3803, 1990; Truckenmiller et al., Res. Iimmunol. 140:527-544, 1989; Van de Perre, et al., New Eng. J. Med. 325:593-598, 1991; Varas et al., BioTechniques 11:384-391, 1991; Velpandi et al., J. Virol. 65:4847-4852, 1991; Williams et al., AIDS 4:393-398, 1990; Zacharet al., J. Virol Meth. 33:391-395, 1991; Zack et al. Cell 61:213-222, 1990; Findlay et al., entitled xe2x80x9cNucleic acid test article and its use to detect a predetermined nucleic acid,xe2x80x9d International Application No. PCT/US90/00452 and International Publication No. WO 90/08840; Gingeras et al., entitled xe2x80x9cNucleic acid probe assay methods and compositions,xe2x80x9d International Application No. PCT/US87/01966 and International Publication No. WO 88/01302; Brakel and Spadoro, entitled xe2x80x9cAmplification capture assay,xe2x80x9d European Patent Application No. 90124738.7 and European Publication No. 0 435 150 A2; Moncany and Montagnier, entitled xe2x80x9cSxc3xa9quences nuclxc3xa9otidiques issues du gxc3xa9nome des rxc3xa9trovirus du typ hiv-1, hiv-2 et siv, et leurs applications notamment pour l""amplification des gxc3xa9nomes de ces rxc3xa9trovirus et pour le diagnostic in-vitro des infections dues xc3xa1 ces virus,xe2x80x9d European Patent Application No. 90401520.3 and European Publication No. 0 403 333 A2; Urdea, entitled xe2x80x9cDNA-dependent RNA polymerase transcripts as reporter molecules for signal amplification in nucleic acid hybridization assays,xe2x80x9d International Application No. PCT/US91/00213 and International Publication No. WO 91/10746; Musso et al., entitled xe2x80x9cLanthanide chelate-tagged nucleic acid probes,xe2x80x9d International Application No. PCT/US88/03735 and International Publication No. WO 89/04375; Chang, entitled xe2x80x9cCloning and expression of HTLV-III DNA,xe2x80x9d European Patent Application No. 85307260.1 and European Publication No. 0 185 444 A2; and Levenson, entitled xe2x80x9cDiagnostic kit and method using a solid phase capture means for detecting nucleic acids,xe2x80x9d European Patent Application No. 89311862.0 and European Publication No. 0 370 694 A2; and Sninsky et al, U.S. Pat. No. 5,008,182.
This invention discloses novel amplification oligonucleotides and detection probes for the detection of Human Immunodeficiency Virus Type 1. The probes are capable of distinguishing between the Human Immunodeficiency Virus type 1 and its known closest phylogenetic neighbors. The amplification oligonucleotides and probes may be used in an assay for the detection and/or quantitation of Human Immunodeficiency Virus nucleic acid.
It is known that a nucleic acid sequence able to hybridize to a nucleic acid sequence sought to be detected (xe2x80x9ctarget sequencexe2x80x9d) can serve as a probe for the target sequence. The probe may be labelled with a detectable moiety such as a radioisotope, antigen or chemiluminescent moiety to facilitate detection of the target sequence. A background description of the use of nucleic acid hybridization as a procedure for the detection of particular nucleic acid sequences is provided in Kohne, U.S. Pat. No. 4,851,330, and Hogan et al., International Application No. PCT/US87/03009 and International Publication No. WO 88/03957, entitled xe2x80x9cNucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organisms.xe2x80x9d
It is also known that hybridization may occur between complementary nucleic acid strands including; DNA/DNA, DNA/RNA, and RNA/RNA. Two single strands of deoxyribo-(xe2x80x9cDNAxe2x80x9d) or ribo-(xe2x80x9cRNAxe2x80x9d) nucleic acid, formed from nucleotides (including the bases adenine (A), cytosine (C), thymidine (T), guanine (G), uracil (U), or inosine (I)), may hybridize to form a double-stranded structure in which the two strands are held together by hydrogen bonds between pairs of complementary bases. Generally, A is hydrogen bonded to T or U, while G is hydrogen bonded to C. At any point along the hybridized strands, therefore, one may find the classical base pairs AT or AU, TA or UA, GC, or CG. Thus, when a first single strand of nucleic acid contains sufficient contiguous complementary bases to a second, and those two strands are brought together under conditions which will promote their hybridization, double-stranded nucleic acid will result. Under appropriate conditions, DNA/DNA, RNA/DNA, or RNA/RNA hybrids may be formed. The present invention includes the use of probes or primers containing nucleotides differing in the sugar moiety, or otherwise chemically modified, which are able to hydrogen bond along the lines described above.
Thus, in a first aspect, the invention features hybridization assay probes able to distinguish Human Immunodeficiency Virus type 1 from other viruses found in human blood or tissues, and amplification oligonucleotides able to selectively amplify Human Immunodeficiency Virus nucleic acid. Specifically, the probes are nucleotide polymers which hybridize to the nucleic acid region of Human Immunodeficiency Virus type 1 corresponding to bases 763-793 of HIV type 1, (HXB2 isolate GenBank accession number K03455), or any of the regions corresponding to bases 1271-1301, 1358-1387, 1464-1489, 1501-1540, 1813-1845, 2969-2999, 3125-3161, 4148-4170, 4804-4832, 5950-5978, 9496-9523, 510-542, and 624-651; preferably, the oligonucleotide comprises, consists essentially of, or consists of the sequence (reading 5xe2x80x2 to 3xe2x80x2)
or RNA equivalents thereto (SEQ. ID. Nos. 67-80), or oligonucleotides complementary thereto (SEQ. ID. Nos. 53-66), or RNA equivalents to the oligonucleotides complementary thereto (SEQ. ID. Nos. 81-94).
The oligonucleotides are used with or without a helper probe as described below. The use of helper probes (e.g., SEQ. ID Nos. 15-18) and complementary oligonucleotides to the helper probes (e.g., SEQ. ID. Nos. 95-98) and RNA equivalents thereto (e.g, SEQ. ID. Nos. 132-139) enhances nucleic acid hybridization.
By xe2x80x9cconsists essentially ofxe2x80x9d is meant that the probe is provided as a purified nucleic acid which under stringent hybridizing conditions hybridizes with the target sequence and not with other related target sequences present in either other virus nucleic acids or human nucleic acids. Such a probe may be linked to other nucleic acids which do not affect such hybridization. Generally, it is preferred that the probe is between 15 to 100 (most preferably between 20 and 50) bases in size. It may, however, be provided in a vector.
In a related aspect, the invention features the formation of nucleic acid hybrids formed by the hybridization of the probes of this invention with target nucleic acid under stringent hybridization conditions. Stringent hybridization conditions involve the use 0.05 M lithium succinate buffer containing 0.6 M LiCl at 60xc2x0 C. The hybrids are useful because they allow the specific detection of viral nucleic acid.
In another related aspect, the invention features amplification oligonucleotides useful for specific detection of Human Immunodeficiency Virus type 1 in an amplification assay. The amplification oligonucleotides are complementary to conserved regions of HIV genomic nucleic acid and are nucleotide polymers able to hybridize to regions of the nucleic acid of HIV corresponding to HIV-1 HXB2R bases 682-705, 800-822, 1307-1337, 1306-1330, 1315-1340, 1395-1425, 1510-1535, 1549-1572, 1743-1771, 1972-1989, 2868-2889, 3008-3042, 3092-3124, 3209-3235, 4052-4079, 4176-4209, 4169-4206, 4394-4428, 4756-4778, 4835-4857, 4952-4969, 5834-5860, 5979-5999, 9431-9457, 9529-9555, 449-473, 550-577, 578-601, 579-600, 624-646, and 680-703.
Specifically, such amplification oligonucleotides consist, comprise, or consist essentially of those selected from (reading 5xe2x80x2 to 3xe2x80x2):
and the RNA equivalents thereto (SEQ. ID. Nos. 99-131). Where (X) is nothing or a 5xe2x80x2 oligonucleotide sequence that is recognized by an enzyme, including but not limited to the promoter sequence for T7, T3, or SP6 RNA polymerase, which enhances initiation or elongation of RNA transcription by an RNA polymerase. One example of X includes the sequence SEQ. ID. NO. 52: 5xe2x80x2-AATTTAATACGACTCACTATAGGGAGA-3xe2x80x2.
These amplification oligonucleotides are used in a nucleic acid amplification assay such as the polymerase chain reaction or an amplification reaction using RNA polymerase, DNA polymerase and RNase H or its equivalent, as described by Kacian and Fultz, U.S. Pat. No. 5,480,784, entitled xe2x80x9cNucleic Acid Sequence Amplification Methods,xe2x80x9d and by Sninsky et al. U.S. Pat. No. 5,079,351, both hereby incorporated by reference herein.
The amplification oligonucleotides and probes of this invention offer a rapid, non-subjective method of identification and quantitation of a sample for specific sequences unique to strains of Human Immunodeficiency Virus type 1.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
We have discovered particularly useful DNA probes complementary to particular nucleic acid sequences of Human Immunodeficiency Virus type 1. Furthermore, we have successfully used these probes in a specific assay for the detection of Human Immunodeficiency Virus type 1, distinguishing it from the known and presumably most closely related taxonomic or phylogenetic neighbors found in human blood or tissues.
We have also identified particularly useful amplification oligonucleotides which are complementary to the Human Immunodeficiency Virus type 1 nucleic acid, and have used these oligonucleotides, e.g., as primers or promoter primer combinations (i.e., a primer having a promoter sequence attached), to amplify the nucleic acid of Human Immunodeficiency Virus, allowing its direct detection in a sample.
Useful guidelines for designing amplification oligonucleotides and probes with desired characteristics are described herein. The optimal sites for amplifying and probing contain two, and preferably three, conserved regions greater than about 15 bases in length, within about 350 bases, and preferably within 150 bases, of contiguous sequence. The degree of amplification observed with a set of primers or promotor/primers depends on several factors, including the ability of the oligonucleotides to hybridize to their complementary sequences and their ability to be extended enzymatically. Because the extent and specificity of hybridization reaction are affected by a number of factors, manipulation of those factors will determine the exact sensitivity and specificity of a particular oligonucleotide, whether perfectly complementary to its target or not. The importance and effect of various assay conditions are described in Hogan et al., International Application No. PCT/US87/03009 and International Publication No. WO 88/03957, entitled xe2x80x9cNucleic Acid Probes for Detection and/or Quantitation of Non-Viral Organismsxe2x80x9d; and Milliman, entitled xe2x80x9cNucleic Acid Probes to Haemophilus influenzae,xe2x80x9d U.S. Pat. No. 5,472,843 and hereby incorporated by reference herein.
The length of the target nucleic acid sequence and, accordingly, the length of the probe sequence can be important. In some cases, there may be several sequences from a particular region, varying in location and length, which will yield probes with the desired hybridization characteristics. In other cases, one sequence may be significantly better than another which differs merely by a single base. While it is possible for nucleic acids that are not perfectly complementary to hybridize, the longest stretch of perfectly homologous base sequence will normally primarily determine hybrid stability. While oligonucleotide probes of different lengths and base composition may be used, oligonucleotide probes preferred in this invention are between about 10 to 50 bases in length and are sufficiently homologous to the target nucleic acid to hybridize under stringent hybridization conditions. We have found that optimal primers have target-binding regions of 18-38 bases, with a predicted Tm (melting temperature) to target of about 65xc2x0 C.
Amplification oligonucleotides or probes should be positioned so as to minimize the stability of the oligomer:nontarget (i.e., nucleic acid with similar sequence to target nucleic acid) nucleic acid hybrid. It is preferred that the amplification oligomers and detection probes are able to distinguish between target and non-target sequences. In designing probes, the differences in these Tm values should be as large as possible (e.g., at least 2xc2x0 C. and preferably 5xc2x0 C.).
Regions of the nucleic acid which are known to form strong internal structures inhibitory to hybridization are less preferred. Examples of such structures include hairpin loops. Likewise, probes with extensive self-complementarity should be avoided.
The degree of non-specific extension (primer-dimer or non-target copying) can also affect amplification efficiency, therefore primers are selected to have low self- or cross-complementarity, particularly at the 3xe2x80x2 ends of the sequence. Long homopolymer tracts and high GC content are avoided to reduce spurious primer extension. Commercial computer programs are available to aid in this aspect of the design. Available computer programs include MacDNASIS(trademark) 2.0 (Hitachi Software Engineering American Ltd.) and OLIGO(copyright) ver. 4.1 (National Bioscience).
Hybridization is the association of two single strands of complementary nucleic acid to form a hydrogen bonded double strand. It is implicit that if one of the two strands is wholly or partially involved in a hybrid that it will be less able to participate in formation of a new hybrid. By designing a probe so that a substantial portion of the sequence of interest is single stranded, the rate and extent of hybridization may be greatly increased. If the target is an integrated genomic sequence then it will naturally occur in a double stranded form, as is the case with the product of the polymerase chain reaction (PCR). These double-stranded targets are naturally inhibitory to hybridization with a probe and require denaturation prior to the hybridization step. Finally, there can be intramolecular and intermolecular hybrids formed within a probe if there is sufficient self complementarity. Such structures can be avoided through careful probe design. Commercial computer programs are available to search for this type of interaction. Available computer programs include MacDNASIS(trademark) 2.0 (Hitachi Software Engineering American Ltd.) and OLIGO(copyright) ver. 4.1 (National Bioscience).
Once synthesized, selected oligonucleotide probes may be labelled by any of several well known methods. 2 J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning 11 (2d ed. 1989). Useful labels include radio-isotopes as well as non-radioactive reporting groups. We currently prefer to use acridinium esters.
Oligonucleotide/target hybrid melting temperature may be determined by isotopic methods well known to those skilled in the art. It should be noted that the Tm for a given hybrid will vary depending on the hybridization solution being used. Sambrook, et al. supra.
Rate of hybridization may be measured by determining the Cot1/2. The rate at which a probe hybridizes to its target is a measure of the thermal stability of the target secondary structure in the probe region. The standard measurement of hybridization rate is the Cot1/2 which is measured as moles of nucleotide per liter times seconds. Thus, it is the concentration of probe times the time at which 50% of maximal hybridization occurs at that concentration. This value is determined by hybridizing various amounts of probe to a constant amount of target for a fixed time. The Cot1/2 is found graphically by standard procedure.
The following examples set forth oligonucleotide probes complementary to a unique nucleic acid sequence from a target organism, and their use in a hybridization assay.